Three hypotheses as to the mechanism of action of murine leukemia viruses (MuLVs) will be tested. I. If MuLVs integrate their proviruses in front of potentially oncogenic cellular sequences, transcription initiated within the provirus could continue into these sequences, increasing their expression, resulting in oncogenic transformation of the cell. Three kinds of experiments will test this hypothesis. (a) RNA from MuMV induced lymphomas will be examined by gel electrophoresis and hybridization to determine if transcription from proviruses extends into cellular sequences. (b) Both RNA and DNA from MuLV induced lymphomas will be analyzed by gel electrophoresis and hydridization to acute oncognic retrovirus probes to determine if integration near and transcription f the cellular genes of known oncogenic potential carried by the acute viruses occurs. Non-viral leukemias will also be examined for increased expression of these sequences. (c) Recombinant DNA techniques will be used to isolate all proviruses and their adjacent cellular sequences from one MuLV induced lymphoma. The adjacent cellular sequences will be used as probes in gel electrophoresis-hybridization experiments to determine if proviruses are integrated near one of these four cellular sites in other MuLV induced lymphomas. II. During their growth in the animal, MuLVs might give rise to variant viruses which are the leukemogenic agents. The viral portion of the four cloned proviruses (described above) will be analyzed by the S1 nuclease-hybridization technique for the variants. III. MuLVs might stimulate target cell division, increasing the probability of a non-viral, oncogenic, cellular mutation. DNA transfer (transfection) experiments will be used to detect any such mutations. Non-viral leukemias will also be examined for oncogenic mutations by transfection experiments. Any oncogenic mutations found in such experiments will be isolated using recombinant DNA technology. A long term goal of this laboratory is, using such clones, to analyze them biologically and biochemically to determine the molecular nature of the events which lead to leukemia development.